Potential Plant-Based New Antiplasmodial Agent Used in Papua Island, Indonesia

Resistance to antimalarial medicine remains a threat to the global effort for malaria eradication. The World Health Organization recently reported that artemisinin partial resistance, which was defined as delayed parasite clearance, was detected in Southeast Asia, particularly in the Greater Mekong subregion, and in Africa, particularly in Rwanda and Uganda. Therefore, the discovery of a potential new drug is important to overcome emerging drug resistance. Natural products have played an important role in drug development over the centuries, including the development of antimalarial drugs, with most of it influenced by traditional use. Recent research on traditional medicine used as an antimalarial treatment on Papua Island, Indonesia, reported that 72 plant species have been used as traditional medicine, with Alstonia scholaris, Carica papaya, Andrographis paniculata, and Physalis minima as the most frequently used medicinal plants. This review aimed to highlight the current research status of these plants for potential novel antiplasmodial development. In conclusion, A. paniculata has the highest potential to be developed as an antiplasmodial, and its extract and known bioactive isolate andrographolide posed strong activity both in vitro and in vivo. A. scholaris and C. papaya also have the potential to be further investigated as both have good potential for their antiplasmodial activities in vivo. However, P. minima is a less studied medicinal plant; nevertheless, it opens the opportunity to explore the potential of this plant.


Introduction
Currently, malaria remains the most severe parasitic disease globally. In 2019, the World Health Organization (WHO) estimated there had been 227 million cases and 558,000 deaths from malaria worldwide. In 2020, the number rose to approximately 241 million cases and 667,000 deaths after the COVID-19 pandemic disrupted services [1]. The reported global incidence rate reduced between 2000 and 2019. However, from 2015 to 2019, the change in the incidence slowed dramatically, and mortality rate reduction was reported to be slower in 2015-2019 [2]. The South-East Asia (SEA) region ranks second behind the African Region in the estimated number of malaria cases, and Indonesia is one of nine malaria-endemic countries in the SEA. The malaria elimination goal in Indonesia must be achieved by 2030 [2,3]. In 2021, the Indonesian Ministry of Health released the Indonesian Health Profile. The Annual Parasite Incidence (API) at the national level was at 0.94 per 1000 people. However, the Papua and Papua Barat provinces still record significantly higher numbers than national values, with 63.12 and 10.15 per 1000 people, respectively, and 95-99% received artemisinin combination treatment (ACT) [4].
Resistance to antimalarial medicine remains a threat to the global effort in malaria eradication. On the way to eliminate malaria, resistance to several drugs such as quinine,

Alstonia scholaris (L.) R. Br.
A. scholaris of the Apocynaceae family is a large, buttressed, evergreen tree that is 6-10 m in height ( Figure 2). It is widespread in the tropical parts of Asia and Africa [15,16]. It is commonly known as the Devil's tree, milkwood pine, and white cheese wood [15]. In Indonesia, it is known as pulai and locally in Papua Island, it is known as arasue, pohon susu, kayu susu, igeii, aikahahe, ibuong, and yepaa [14].

Ethnopharmacology
In various traditional medicines, A. scholaris has been used as and is considered an important medicinal plant. It is commonly used to treat fever, diarrhea, malaria, dysentery, dyspepsia, asthma, bronchitis, as an antidiabetic, etc. [20,21]. In the Indian traditional medicine system, Ayurveda, the bark is used as a tonic, alterative, febrifuge, and gastrointestinal sedative. It is also useful in the treatment of malarial fever [22]. In traditional Chinese medicine, the leaf is used to treat chronic respiratory diseases [23]. In Indonesia, mainly on Papua Island, the bark is used to treat fever and malaria. According to the Papua Health Department, local people use dried bark. It was soaked overnight and one glass is consumed daily to combat malaria, until the disease is cured. Alternatively, fresh bark is chewed directly, and then the liquid or juice is swallowed. However, how much bark should be used as medication has not been mentioned [14,24].

Antiplasmodial Activity
Several in vitro and in vivo studies have investigated the antiplasmodial activities of A. scholaris. Two in vitro studies on the chloroquine-sensitive P. falciparum strain 3D7 were performed by Taek et al. (2021) and Abdillah et al. (2015) who investigated the activity of stem bark extract. The only difference in their method was the solvent used in the extraction by maceration, which were 95% and 70% ethanol. However, a quite significant difference was found in the IC 50 result, where extraction with 70% ethanol gives a strong antiplasmodial activity (0.165 µg/mL) compared to 95% ethanol with only moderate activity (15.46 µg/mL) [43,44] Keawpradub et al. (1999) compared the in vitro antiplasmodial activity against the P. falciparum multidrug-resistant K1 strain from a methanolic extract using leaves, stem bark, and root bark of three Alstonia species from Thailand. The result showed that the stem bark of A. scholaris was second best with the lowest IC 50 of 181.4 µg/mL, which is considered inactive, much weaker than the root bark of Alstonia macrophylla with the lowest IC 50 of 5.7 µg/mL [45,46]. This result suggested that the methanolic extract of A. scholaris might not well deal with the P. falciparum multidrug-resistant strain, although other studies on the chloroquine-sensitive strain gave a strong and moderate activity [43,44]. This study also tested villalstonine and macrocarpamine which were isolated from A. macrophylla and well known to be present in A. scholaris. Interestingly, these isolates gave a strong activity with IC 50 of 0.27 and 0.36 µg/mL, respectively. This could indicate that A. macrophylla might contain higher levels of villalstonine and macrocarpamine ( Figure 3) than A. scholaris, although further research is needed for confirmation. The author suggested that these active alkaloids might act through a different mechanism to chloroquine and further investigation is needed to provide evidence [46]. The possible mechanism of bisindole alkaloids is the inhibition of hemozoin formation but in a manner different from chloroquine; hence, this explains the activity against chloroquine-resistant P. falciparum [47].
further in vivo study. The in vitro and in vivo studies of A. scholaris are summarized in Table 1.
The toxicity profile of A. scholaris has been studied for its methanolic and hydroalcoholic extract. Bello et al. (2016) conducted the acute and subacute toxicity studies of methanolic extract in Sprague-Dawley rats. In the acute study, the extract, at a dose of 2000 mg/kg BW, showed no toxicity, whereas in the subacute study, two female rats were recorded to have died at the highest dose of 1000 mg/kg BW after 28 days of oral administration. In histopathological studies, liver damage was found at the long-term administration of a high dose [50]. Baliga et al. (2004) investigated the acute and subacute toxicity of the hydroalcoholic extract of A. scholaris in mice and rats. In an acute study, the oral administration of 2000 mg/kg BW to Swiss albino mice showed no toxicity, whereas 1100 mg/Kg BW administered intraperitoneally resulted in animal deaths. The subacute toxicity test showed 30% mortality at a dose of 240 mg/kg BW given intraperitoneally, with damage in all organs [51]. From this study, oral administration of A. scholaris methanolic and hydroalcoholic extracts, as the administration route of the activity studies, showed low toxicity according to the OECD 425 guidelines [52].  The in vivo activity of A. scholaris stem bark has also been studied. Abdullah et al. (2015) continued their previous in vitro study against the Plasmodium bergheii NK 65 strain in mice. The ethanolic extract of A. scholaris stem bark gave an ED 50 of 121.94 mg/kg which was categorized as good activity [44]. According to Muñoz et al. (2000), in vivo antiplasmodial activity is classified as excellent if ED 50 is ≤100 mg/kg BW/day, good if ED 50 is ≤101-250 mg/kg BW/day, moderate if ED 50 is 251-500 mg/kg BW/day, and inactive if ED 50 is >500 mg/kg BW/day [48]. In addition, in an in vivo study, Intan et al. investigate the activity of a mixed extract of A. scholaris and Phyllanthus niruri (1:1). The extract was obtained by maceration with 70% ethanol, which reduced parasitemia and differential leukocyte count at a dose of 147.78 mg/kg [49]. Both studies have shown that the 70% ethanol extract of A. scholaris exerted a good in vivo antiplasmodial effect, although the second study used a mixture of two plant extracts. From the studies discussed, stem bark ethanolic extract should be the focus of further investigation against the P. falciparum chloroquine-resistant strain and the isolate, villalstonine, and macrocarpamine should be subjected to further in vivo study. The in vitro and in vivo studies of A. scholaris are summarized in Table 1.
The toxicity profile of A. scholaris has been studied for its methanolic and hydroalcoholic extract. Bello et al. (2016) conducted the acute and subacute toxicity studies of methanolic extract in Sprague-Dawley rats. In the acute study, the extract, at a dose of 2000 mg/kg BW, showed no toxicity, whereas in the subacute study, two female rats were recorded to have died at the highest dose of 1000 mg/kg BW after 28 days of oral administration. In histopathological studies, liver damage was found at the long-term administration of a high dose [50]. Baliga et al. (2004) investigated the acute and subacute toxicity of the hydroalcoholic extract of A. scholaris in mice and rats. In an acute study, the oral administration of 2000 mg/kg BW to Swiss albino mice showed no toxicity, whereas 1100 mg/Kg BW administered intraperitoneally resulted in animal deaths. The subacute toxicity test showed 30% mortality at a dose of 240 mg/kg BW given intraperitoneally, with damage in all organs [51]. From this study, oral administration of A. scholaris methanolic and hydroalcoholic extracts, as the administration route of the activity studies, showed low toxicity according to the OECD 425 guidelines [52].

Carica papaya L.
C. papaya of the Caricaceae family is a tree or shrub 8-10 m in height ( Figure 4). It is native to tropical America but is cultivated all over the tropics and subtropics [70]. It is traditionally widely used as a treatment. In Indonesia, it is generally known as Pepaya. In Papua Island it is also locally known as peceren, eteradado, monofo, papaya, labseren, ogoseren, cacaveka, mbikin, senene ranau, and pepaya bllah [14]. C. papaya of the Caricaceae family is a tree or shrub 8-10 m in height ( Figure 4). It is native to tropical America but is cultivated all over the tropics and subtropics [70]. It is traditionally widely used as a treatment. In Indonesia, it is generally known as Pepaya. In Papua Island it is also locally known as peceren, eteradado, monofo, papaya, labseren, ogoseren, cacaveka, mbikin, senene ranau, and pepaya bllah [14].

Ethnopharmacology
C. papaya is used as a treatment for various conditions. Its leaf is used to treat dengue, inhibit cancer growth, treat malaria, for digestion problems and acne, increased appetite, to ease menstrual pain, relieve nausea, jaundice, kill viruses, and modulate immune response [14,74]. The fruit is used as a laxative and a treatment for indigestion [74]. The seed is used as a contraceptive for men and for abortion in women [75]. The flowers are utilized as digestive disorder treatments, diuretics, and tonics. In Papua Island, C. papaya leaf is used to combat malaria. In Waibon village, locals squeeze the yellowish leaves, add a pinch of salt, and consume the mixture orally. In other villages, young leaves are boiled with three glasses of water until reduced to one glass and then consumed orally [14,76].

Antiplasmodial Activity
Antiplasmodial studies on different parts of C. papaya have been performed. Three studies used different extraction methods and examined the in vitro antiplasmodial activity of leaves. used maceration, percolation, and soxhlet extraction methods, respectively, with ethanol as solvent, and all were tested against P. falciparum. The IC50 of the ethanolic extract by maceration, percolation, and soxhlet methods were 0.18, 46.23, and 25 µ g/mL, respectively [44,53,60]. Results varied, as the activity of the ethanolic extract obtained with the maceration method was classified as strong, while those with percolation and Soxhlet methods were classified as moderate [45]. A noticeable difference was due to the P. falciparum strain used. Abdillah

Ethnopharmacology
C. papaya is used as a treatment for various conditions. Its leaf is used to treat dengue, inhibit cancer growth, treat malaria, for digestion problems and acne, increased appetite, to ease menstrual pain, relieve nausea, jaundice, kill viruses, and modulate immune response [14,74]. The fruit is used as a laxative and a treatment for indigestion [74]. The seed is used as a contraceptive for men and for abortion in women [75]. The flowers are utilized as digestive disorder treatments, diuretics, and tonics. In Papua Island, C. papaya leaf is used to combat malaria. In Waibon village, locals squeeze the yellowish leaves, add a pinch of salt, and consume the mixture orally. In other villages, young leaves are boiled with three glasses of water until reduced to one glass and then consumed orally [14,76].

Phytochemistry
Various parts of C. papaya are reported to contain caffeic acid, myricetin, quercetin, papain, α-tocopherol, benzyl isothiocyanate, kaempferol, squalene, phytol, campesterol, stigmasterol, β-sitosterol, hexadecanoid acid, γ-tocopherol, olean-12-ene, 13,17-cyclocursan-3-one, cycloartenol, and carpaine [77,78]. Another study characterized C. papaya seed oil and reported that it contains oleic, palmitic, stearic, linoleic, palmitoleic, arachidic, gadoleic, myristic, and margoric acid [79]. used maceration, percolation, and soxhlet extraction methods, respectively, with ethanol as solvent, and all were tested against P. falciparum. The IC 50 of the ethanolic extract by maceration, percolation, and soxhlet methods were 0.18, 46.23, and 25 µg/mL, respectively [44,53,60]. Results varied, as the activity of the ethanolic extract obtained with the maceration method was classified as strong, while those with percolation and Soxhlet methods were classified as moderate [45]. A noticeable difference was due to the P. falciparum Moreover, the stem was also subjected to in vitro testing by Ravikumar et al. using percolation extraction with ethanol extract; however, it demonstrated less activity with an IC 50 of 65.13 µg/mL [53]. In addition, the in vitro activity in C. papaya leaves was investigated by Teng et al. (2019), who reported that the alkaloidal n-hexane extract was the most active against P. falciparum 3D7 and Dd2 strains with IC 50 of 3.45 and 1.52 µg/mL, followed by dichloromethane extract with IC 50 of 7.67 and 4.50 µg/mL, respectively. Carpaine ( Figure 5A) was isolated from alkaloidal n-hexane extract and further screened against the P. falciparum 3D7 strain. It showed better activity with an IC 50 of 2.01 µg/mL, suggesting the bioactive compound from the leaves [57]. Julianti et al. (2014) also isolated and tested carpaine in vitro against P. falciparum, which showed strong activity with an IC 50 of 0.2 µM [56]. Melariri et al. (2011) tested several extracts obtained by sequential extraction and showed that the ethyl acetate extract was the most active with 2.6 µg/mL against P. falciparum chloroquine-sensitive D10 strain. Two isolates, linolenic acid and linoleic acid ( Figure 5B,C) were then obtained and tested, which gave strong and good activity against P. falciparum chloroquine-resistant with IC 50 of 3.58 and 6.88 µg/mL, respectively [55]. A previous study reported that fatty acids could enhance the neutrophil-mediated killing of the asexual blood forms of P. falciparum and that they might be a possible mechanism for linolenic and linonelic acid activity, although further confirmation is needed [80].

Antiplasmodial Activity
x FOR PEER REVIEW 11 of 21 were 400 mg/Kg BW. Petroleum ether, chloroform, and methanol extracts of fruit rind showed parasite suppression rates of 61.78%, 37.65%, and 18.39%, and extracts from roots had rates of 43.77%, 48.11%, and 25.63%, respectively. Only petroleum ether extract achieved a parasite suppression rate of >50%, which was classified as good [48,58]. Based on all these studies, the fruit rind and roots of C. papaya likely have less potential as antiplasmodial agents compared with leaves as summarized in Table 1. The antiplasmodial mechanism is still unclear; however, macrocylic dilactone alkaloids such as carpaine might work as a cation-chelating agent based on their structure [81]. From these studies, a direct comparison between methanolic, ethanolic, and ethyl acetate extracts might be performed against the P. falciparum chloroquine-resistant strain as the result from studies available varies. Moreover, the constituents of the most active extract should be determined to investigate the possibility of its synergistic action. Further in vivo studies must confirm the in vitro activity of the extracts and linolenic acid, which posed a strong activity in vitro against both P. falciparum chloroquine-sensitive and resistant strains. The animal toxicity study of C. papaya leaf was summarized by Lim et al. (2021) in a systematic scoping review where, generally, leaf juice and aqueous extracts at doses up to 2000 mg/Kg are nontoxic in rats in acute and subacute studies [82][83][84]. The hydroalcoholic extract also showed very low toxicity with an LD50 of 5120 mg/kg BW when administered orally in chicks and no toxicity was noted in oral subchronic administration at a low dose [85]. The methanol sub-fraction from the chloroform extract of seeds showed no developmental toxicity or teratogenicity at the highest dose of 500 mg/kg BW [86]. The clinical evidence also suggests that oral consumption of C. papaya leaf, which is mostly aqueous extract or juice, is well tolerated [84].

Andrographis paniculata (Burm. f.)
A. paniculata of the Acanthaceae family is an herb that is up to 50 cm tall, annual, branched, and extremely bitter in all parts of the plant body ( Figure 6) [87,88]. It is native to Taiwan, mainland China, India and Sri Lanka, is widely found in southern and southeast Asia, such as Bangladesh, China, Indonesia, Malaysia, Myanmar, Philippines, and Thailand [88][89][90][91]. It is commonly known as the king of bitter or kalmegh and, in Indonesia, it is known as sambiloto, sambiroto, and samiroto [14].  [54,59]. Munoz et al. also stated that in vivo antiplasmodial activity can be classified as moderate, good, and very good if an extract suppressed parasitemia by >50% at doses of 500, 250, and 100 mg/kg per BW/day, respectively [48]. Based on that classification, the activities of methanolic and aqueous extract were considered very good and moderate, respectively. However, Okpe et al. only tested for one dose at a time, and the parasite suppression result was on par with the positive control (halofantrine 25 mg/kg BW); therefore, it is still possible to achieve parasite suppression ≥50% with a lower dose [59]. In addition, Julianti et al.
(2014) investigated the carpaine isolate for its in vivo antiplasmodial activity; however, the results were somewhat disappointing as the treatment only reduced parasitemia by 11.9% despite the promising result of an in vitro study [56]. However, the exact dose was not mentioned; thus, we cannot conclude whether the low activity was due to the low dose given or other factors. From these studies, C. papaya leaf extract showed good potential as an antiplasmodial agent; however, the active compound must be further studied given the contrasting results of Carpaine in vitro and in vivo. In addition, the possibility of a synergistic effect of the compounds contained in the extract should also be considered.
Other candidates as bioactive compounds should be considered and further examined because strong in vitro activity against chloroquine-resistant P. falciparum was found.
The fruit rind and roots of C. papaya were also studied for their in vivo antiplasmodial activities. In this study, Zeleke et al. (2017) obtained extracts through the Soxhlet method using three solvents, namely petroleum ether, chloroform, and methanol. The doses used were 400 mg/Kg BW. Petroleum ether, chloroform, and methanol extracts of fruit rind showed parasite suppression rates of 61.78%, 37.65%, and 18.39%, and extracts from roots had rates of 43.77%, 48.11%, and 25.63%, respectively. Only petroleum ether extract achieved a parasite suppression rate of >50%, which was classified as good [48,58]. Based on all these studies, the fruit rind and roots of C. papaya likely have less potential as antiplasmodial agents compared with leaves as summarized in Table 1. The antiplasmodial mechanism is still unclear; however, macrocylic dilactone alkaloids such as carpaine might work as a cation-chelating agent based on their structure [81]. From these studies, a direct comparison between methanolic, ethanolic, and ethyl acetate extracts might be performed against the P. falciparum chloroquine-resistant strain as the result from studies available varies. Moreover, the constituents of the most active extract should be determined to investigate the possibility of its synergistic action. Further in vivo studies must confirm the in vitro activity of the extracts and linolenic acid, which posed a strong activity in vitro against both P. falciparum chloroquine-sensitive and resistant strains.
The animal toxicity study of C. papaya leaf was summarized by Lim et al. (2021) in a systematic scoping review where, generally, leaf juice and aqueous extracts at doses up to 2000 mg/Kg are nontoxic in rats in acute and subacute studies [82][83][84]. The hydroalcoholic extract also showed very low toxicity with an LD 50 of 5120 mg/kg BW when administered orally in chicks and no toxicity was noted in oral subchronic administration at a low dose [85]. The methanol sub-fraction from the chloroform extract of seeds showed no developmental toxicity or teratogenicity at the highest dose of 500 mg/kg BW [86]. The clinical evidence also suggests that oral consumption of C. papaya leaf, which is mostly aqueous extract or juice, is well tolerated [84].

Andrographis paniculata (Burm. f.)
A. paniculata of the Acanthaceae family is an herb that is up to 50 cm tall, annual, branched, and extremely bitter in all parts of the plant body ( Figure 6) [87,88]. It is native to Taiwan, mainland China, India and Sri Lanka, is widely found in southern and southeast Asia, such as Bangladesh, China, Indonesia, Malaysia, Myanmar, Philippines, and Thailand [88][89][90][91]. It is commonly known as the king of bitter or kalmegh and, in Indonesia, it is known as sambiloto, sambiroto, and samiroto [14]. lants 2023, 12, x FOR PEER REVIEW 12 of 21

Ethnopharmacology
Commonly, aerial parts of A. paniculata are used to treat various infectious diseases such as leprosy, gonorrhea, respiratory tract infections, scabies, malaria, helminthiasis, herpes, peptic ulcers, and is used topically for skin infection and snake bites. It is also used as a treatment for irregular bowel habits, loss of appetite, alopecia, diabetes, jaundice, dyspepsia, and coughs [88][89][90][91]94]. In China, it was described as bitter and cold and considered antipyretic, detoxicant, anti-inflammatory, and detumescent. It is also thought to be used to remove "pathogenic heat" from the blood [90]. In Papua, A. paniculata is used to combat malaria. They use 10-15 leaves of sambiloto, boil them with water, and drink them two times a day until the disease is cured [24].

Antiplasmodial Activity
Among these four plants, A. paniculata is the most well studied for its antiplasmodial activity. In vitro studies were conducted by three research groups. Rahman et al. (1999) performed in vitro testing of the whole A. paniculata plant sequentially extracted by the Soxhlet method with n-hexane, chloroform, and methanol. Only chloroform and methanol extracts were tested and the results showed that the chloroform extract at a dose of 0.05 mg/mL had an inhibition rate of 100% after 24 h and at a dose of 0.025 mg/mL showed 100% inhibition after 48 h. The methanol extract at a dose of 0.4 mg/mL showed 100% inhibition after 24 h and at 0.025 mg/mL showed 100% inhibition after 72 h. However, the IC50 value was not determined [68]. Mishra et al. (2009) investigated the in vitro antiplasmodial activity of aerial parts of A. paniculata, extracted by the maceration method at 4 °C overnight with 98% methanol and tested against two strains of P. falciparum, the chloroquine-sensitive (MRC-pf-20) and choloroquine-resistant (MRC-pf-303). The IC50 was 7.2 µ g/mL which, according to Ouattara et al., was considered strong or very active [45,65]. The results showed that the extract gave good activity for both strains, suggesting that the

Ethnopharmacology
Commonly, aerial parts of A. paniculata are used to treat various infectious diseases such as leprosy, gonorrhea, respiratory tract infections, scabies, malaria, helminthiasis, herpes, peptic ulcers, and is used topically for skin infection and snake bites. It is also used as a treatment for irregular bowel habits, loss of appetite, alopecia, diabetes, jaundice, dyspepsia, and coughs [88][89][90][91]94]. In China, it was described as bitter and cold and considered antipyretic, detoxicant, anti-inflammatory, and detumescent. It is also thought to be used to remove "pathogenic heat" from the blood [90]. In Papua, A. paniculata is used to combat malaria. They use 10-15 leaves of sambiloto, boil them with water, and drink them two times a day until the disease is cured [24].

Antiplasmodial Activity
Among these four plants, A. paniculata is the most well studied for its antiplasmodial activity. In vitro studies were conducted by three research groups. Rahman et al. (1999) performed in vitro testing of the whole A. paniculata plant sequentially extracted by the Soxhlet method with n-hexane, chloroform, and methanol. Only chloroform and methanol extracts were tested and the results showed that the chloroform extract at a dose of 0.05 mg/mL had an inhibition rate of 100% after 24 h and at a dose of 0.025 mg/mL showed 100% inhibition after 48 h. The methanol extract at a dose of 0.4 mg/mL showed 100% inhibition after 24 h and at 0.025 mg/mL showed 100% inhibition after 72 h. However, the IC 50 value was not determined [68]. Mishra et al. (2009) investigated the in vitro antiplasmodial activity of aerial parts of A. paniculata, extracted by the maceration method at 4 • C overnight with 98% methanol and tested against two strains of P. falciparum, the chloroquine-sensitive (MRC-pf-20) and choloroquine-resistant (MRC-pf-303). The IC 50 was 7.2 µg/mL which, according to Ouattara et al., was considered strong or very active [45,65]. The results showed that the extract gave good activity for both strains, suggesting that the extract could well deal with the chloroquine-resistance mechanism of P. falciparum [65]. In another study, Zein et al. (2013) investigated the in vitro activity of A. paniculata extract through the maceration method for 3 h with 50% ethanol as a solvent against P. falciparum of Papua strain (2300). Accordingly, at a dose of 200 µg/mL, the parasite density was 10% compared with 38.7% in the control. However, this study also did not calculate the IC 50 value [63].
The investigation of the in vivo activity was performed by Rahman et al. (1999), which further tested the methanolic extract for its in vivo antiplasmodial activity against P. berghei. At a dose of 5 mg/kg administered intraperitoneally for 4 days, the extract group showed delayed parasitemia where all mice died on day 7, compared with the control group without treatment where all mice died on day 5 [68]. This result is different to that reported by Mishra et al. (2009), who continued their investigation in vitro to evaluate the in vivo activity of the methanolic extract. Accordingly, at a dose of 7 mg/Kg BW, 39% parasitemia was observed on day 12, suggesting good suppression [65]. These two studies differed in the extraction method used. Sequential extraction by the Soxhlet method with n-hexane, chloroform, and methanol appeared to reduce the bioactive content in methanolic extract in the study by Rahman et al. (1999), compared with direct soaking overnight at 4 • C by Mishra et al. (2009), which appeared to be a better extraction method. In addition, the oral administration route appears to be superior to intraperitoneal administration [65,68]. Another in vivo study was performed by Widyawaruyanti et al. (2014), who used a purified ethyl acetate fraction from a 96% ethanol extract in tablet form. The study showed that 15 mg pure fraction per 300 mg tablet by wet granulation resulted in 78.16% parasite suppression and 60 mg pure fraction per 150 mg tablet by the solid dispersion method achieved 80.35% parasite suppression which, according to Munoz et al., was classified as very good [48,61]. The same research group further conducted a double-blind controlled crossover clinical trial (phase 1) for this pure fraction in 30 healthy volunteers, with a dose equivalent to 35 mg andrographolide per tablet. The study reported no difference in vital signs, hematological, and biochemical parameters when compared with the control group (placebo). In addition, increased appetite and better sleep were reported after the administration of the test tablet, which could be beneficial in uncomplicated malaria in which one of the symptoms is anorexia [62,109]. Hassan et al. (2019) studied the isolate, investigating the in vitro and in vivo activities of andrographolide (Figure 7). The IC 50 of andrographolide against the P. falciparum 3D7 strain was 13.70 µM or roughly 4.8 µg/mL when converted, as the molar mass of andrographolide is 350.45 g/mol which was considered strong. The in vivo activity against P. berghei in infected mice with a dose of 5 mg/kg BW achieved 60.17% parasite suppression and, according to Munoz et al., it is considered very good [45,48,67]. This study is in line with the previous study by Mishra et al. (2011), in which the in vitro activity against the P. falciparum chloroquine-resistant strain was considered strong with an IC 50 of 9.1 µM. The in vivo study against P. berghei at a dose of 15 mg/kg showed parasitemia level at 46% (>50% suppression) on day 13, where all controls died [66]. This shows that andrographolide could be the bioactive compound of A. paniculata demonstrating antiplasmodial activity as shown in Table 1, which has cytokine-modulating effects through the inhibition of GSK3β [67]. Nevertheless, other compounds may also contribute, or their interaction may eventually influence the extract activity. From these studies, the ethanolic extracts and ethyl acetate fraction might be investigated in a broader clinical study to confirm their efficacy for multi-compound product development, and andrographolide appears to be a definite bioactive compound, given its consistency in the in vitro and in vivo studies.
The acute toxicity study from the first true leaf ethanolic extract of A. paniculata in mice showed that a single oral administration with a dose of 5000 mg/Kg BW has no significant acute toxicological effect [110]. Another acute toxicity study showed that the methanolic extract of A. paniculata leaves at a dose of 5000 mg/Kg BW did not pose any treatment-related adverse clinical signs after 14 days of administration [111]. Moreover, the ethanolic extract of the aerial part of the plant did not show any testicular toxicity at a dose of 1000 mg/kg BW after 60 days of forced administration by buco-gastric formula [112]. In the acute toxicity study of its bioactive compound andrographolide, a dose of 500 mg/kg BW orally did not cause any toxicity; hence, it is considered relatively nontoxic [113]. Another study of doses up to 5 g/kg BW orally in mice did not show any signs of acute toxicity, whereas oral administration at a dose of up to 500 mg/Kg BW in Wistar rats for 21 days for subacute toxicity evaluation did not result in death or other signs of toxicity [114]. These studies suggest the relatively nontoxic properties of A. paniculata extracts and andrographolide as its bioactive compound. dose of 1000 mg/kg BW after 60 days of forced administration by buco-gastric formula [112]. In the acute toxicity study of its bioactive compound andrographolide, a dose of 500 mg/kg BW orally did not cause any toxicity; hence, it is considered relatively nontoxic [113]. Another study of doses up to 5 g/kg BW orally in mice did not show any signs of acute toxicity, whereas oral administration at a dose of up to 500 mg/Kg BW in Wistar rats for 21 days for subacute toxicity evaluation did not result in death or other signs of toxicity [114]. These studies suggest the relatively nontoxic properties of A. paniculata extracts and andrographolide as its bioactive compound.

Ethnopharmacology
Traditionally, P. minima is used as an antiulcer, anti-inflammatory, antimalarial, antidiabetic, antimicrobial, diuretic, purgative, and cardioprotective plant [118,120]. The fruits and flowers are used to treat stomach pain and constipation. Herb paste is also applied to ear disorders [121,122]. In the Malay community, the decoction of the whole plant is used to cure cancer [123]. In Papua Island, Indonesia, P. minima is among the top four medicinal plants used to combat malaria; however, no specific usage was explained in the literature [14].

Physalis minima L.
Physalis minima Linn. (Solanaceae) is a small herbaceous annual plant grown as a weed in crop fields, found widely in Indonesia, Malaysia, India, Afghanistan, tropical Africa, and Australia ( Figure 8) [115][116][117][118]. The plant has a bitter taste and is known in Indonesia as ciplukan and, in Papua Island, it is locally known as pakpak, toki, daun nipon, cemploka, and seberi-nembai [14,117,118]. dose of 1000 mg/kg BW after 60 days of forced administration by buco-gastric formula [112]. In the acute toxicity study of its bioactive compound andrographolide, a dose of 500 mg/kg BW orally did not cause any toxicity; hence, it is considered relatively nontoxic [113]. Another study of doses up to 5 g/kg BW orally in mice did not show any signs of acute toxicity, whereas oral administration at a dose of up to 500 mg/Kg BW in Wistar rats for 21 days for subacute toxicity evaluation did not result in death or other signs of toxicity [114]. These studies suggest the relatively nontoxic properties of A. paniculata extracts and andrographolide as its bioactive compound.

Ethnopharmacology
Traditionally, P. minima is used as an antiulcer, anti-inflammatory, antimalarial, antidiabetic, antimicrobial, diuretic, purgative, and cardioprotective plant [118,120]. The fruits and flowers are used to treat stomach pain and constipation. Herb paste is also applied to ear disorders [121,122]. In the Malay community, the decoction of the whole plant is used to cure cancer [123]. In Papua Island, Indonesia, P. minima is among the top four medicinal plants used to combat malaria; however, no specific usage was explained in the literature [14].

Ethnopharmacology
Traditionally, P. minima is used as an antiulcer, anti-inflammatory, antimalarial, antidiabetic, antimicrobial, diuretic, purgative, and cardioprotective plant [118,120]. The fruits and flowers are used to treat stomach pain and constipation. Herb paste is also applied to ear disorders [121,122]. In the Malay community, the decoction of the whole plant is used to cure cancer [123]. In Papua Island, Indonesia, P. minima is among the top four medicinal plants used to combat malaria; however, no specific usage was explained in the literature [14].

Antiplasmodial Activity
To date, P. minima is less studied among the four plants used as antiplasmodial treatments in Papua Island; in fact, we have not found an antiplasmodial or antimalarial study of the plant. However, in Indonesia, P. minima is known as "ciplukan" and another species, Physalis angulate, is also called "ciplukan". Lusakibanza et al. (2010) investigated the in vitro and in vivo antiplasmodial activities of P. angulata, whose methanolic and choloroform extracts demonstrated a strong in vitro activity according to Ouattara et al. against the chloroquine-sensitive P. falciparum strain 3D7 with IC 50 of 1.25 and 1.96 µg/mL, and against the chloroquine-resistant P. falciparum strain W2 with IC 50 of 3.02 and 2.00 µg/mL, respectively. The in vivo study showed that the aqueous extract at a dose of 300 mg/kg BW achieved 58.7% parasite suppression which, according to Munoz et al., is considered good [48,132]. Moreover, Sa et al. (2011) investigated the antiplasmodial properties of physalines B, D, F, and G. These compounds are also present in P. minima. The in vitro study showed that physaline F ( Figure 9B) is the most active, with the lowest IC 50 of 2.2 µg/mL, and physaline D ( Figure 9A) is the less active among them with IC 50 of 55 µg/mL. Interestingly, in the in vivo study, physaline D showed better activity with 65% parasite suppression at a dose of 100 mg/kg BW, which was considered very good according to Munoz et al., compared with physalin F with no parasite suppression at 100 mg/kg BW. Thus, suppression induced by physalin F might inhibit the antimalarial immune response to control infection in infected animals (Table 1); therefore, the better antimalarial activity of physalin D is probably due to its lack of immunosuppressive activity in mice [48,69]. Physalin D inhibits the P2X7 receptor [133]. Plasmodium infection releases large amounts of ATP, which results in sustained P2X7 receptor activation leading to a pro-inflammatory cycle [134]. Thus, treatment with P2 inhibitor that blocks these receptors will impair invasion [135]. This report is a good example of where the strong IC 50 value of the in vitro analysis might not directly reflect a good in vivo activity as a more complex system was involved in the in vivo study, emphasizing the importance of the in vivo study. Given the lack of activity studies, a preliminary activity screening of the extracts is suggested. pounds, namely, 5-methoxy-6,7-methylenedioxyflavone and 5,6,7-trimethoxyflavone [131].

Antiplasmodial Activity
To date, P. minima is less studied among the four plants used as antiplasmodial treatments in Papua Island; in fact, we have not found an antiplasmodial or antimalarial study of the plant. However, in Indonesia, P. minima is known as "ciplukan" and another species, Physalis angulate, is also called "ciplukan". Lusakibanza et al. (2010) investigated the in vitro and in vivo antiplasmodial activities of P. angulata, whose methanolic and choloroform extracts demonstrated a strong in vitro activity according to Ouattara et al. against the chloroquine-sensitive P. falciparum strain 3D7 with IC50 of 1.25 and 1.96 µ g/mL, and against the chloroquine-resistant P. falciparum strain W2 with IC50 of 3.02 and 2.00 µ g/mL, respectively. The in vivo study showed that the aqueous extract at a dose of 300 mg/kg BW achieved 58.7% parasite suppression which, according to Munoz et al., is considered good [48,132]. Moreover, Sa et al. (2011) investigated the antiplasmodial properties of physalines B, D, F, and G. These compounds are also present in P. minima. The in vitro study showed that physaline F ( Figure 9B) is the most active, with the lowest IC50 of 2.2 µ g/mL, and physaline D ( Figure 9A) is the less active among them with IC50 of 55 µ g/mL. Interestingly, in the in vivo study, physaline D showed better activity with 65% parasite suppression at a dose of 100 mg/kg BW, which was considered very good according to Munoz et al., compared with physalin F with no parasite suppression at 100 mg/kg BW. Thus, suppression induced by physalin F might inhibit the antimalarial immune response to control infection in infected animals (Table 1); therefore, the better antimalarial activity of physalin D is probably due to its lack of immunosuppressive activity in mice [48,69]. Physalin D inhibits the P2X7 receptor [133]. Plasmodium infection releases large amounts of ATP, which results in sustained P2X7 receptor activation leading to a pro-inflammatory cycle [134]. Thus, treatment with P2 inhibitor that blocks these receptors will impair invasion [135]. This report is a good example of where the strong IC50 value of the in vitro analysis might not directly reflect a good in vivo activity as a more complex system was involved in the in vivo study, emphasizing the importance of the in vivo study. Given the lack of activity studies, a preliminary activity screening of the extracts is suggested.
In line with its pharmacological activity, toxicological data from P. minima is also limited. The water extract of P. minima leaves did not show any acute signs of toxicity at a dose of 2000 mg/kg BW after 14 days of oral administration [136]. The subchronic toxicity of the aqueous extract at a dose of 450 mg/Kg BW after 90 days of oral administration did not show any signs of hematologic toxicity, hepatotoxicity, or renal toxicity [137]. The lack of toxicity data for other organic solvent extracts such as methanol or ethanol, and its possible bioactive compound, physalins, is a challenge to the development of potential antiplasmodial agents from P. minima and to providing the safety profile.  In line with its pharmacological activity, toxicological data from P. minima is also limited. The water extract of P. minima leaves did not show any acute signs of toxicity at a dose of 2000 mg/kg BW after 14 days of oral administration [136]. The subchronic toxicity of the aqueous extract at a dose of 450 mg/Kg BW after 90 days of oral administration did not show any signs of hematologic toxicity, hepatotoxicity, or renal toxicity [137]. The lack of toxicity data for other organic solvent extracts such as methanol or ethanol, and its possible bioactive compound, physalins, is a challenge to the development of potential antiplasmodial agents from P. minima and to providing the safety profile.

Conclusions
Among these four medicinal plants traditionally used to treat malaria from Papua Island, Indonesia, A. paniculata is the most extensively studied and is a potential medicinal plant for antiplasmodial development, having completed the in vitro, in vivo, phases 1 and 2 of clinical trials, and a toxicity profile. The extract and its bioactive compound, andrographolide, have the potential to be developed as an antiplasmodial agent whether as a multicompound or single-compound product. A. scholaris and C. papaya also have the potential to be further investigated for determining their bioactive compounds as both posed good potential for antiplasmodial activities in vivo and have extensive toxicity data; however, the considered bioactive compounds must be evaluated for their in vivo antiplasmodial activity. P. minima is a less studied medicinal plant among the others; nevertheless, it allows for more potential investigation as this plant is also used extensively in traditional remedies to treat malaria.